Traditional approaches in drug development have focused on the use of therapeutic agents capable of interacting directly with proteins involved in disease states or other states of unhealth. Drugs borne of this tradition include, for example, synthetic hormones (to simulate the function of protein-based hormones desirably present in the body), antibiotics (which attack foreign proteins, namely microorganisms) and vitamins (which provide the building blocks required by certain proteins to perform their ordinary function in the body), in addition to many others. More recently, therapeutic agents in the form of oligonucleotides have been designed to indirectly regulate, control, or otherwise impact protein function by altering at the genetic level the blueprint or machinery that controls synthesis of all proteins. Because each gene contains the information necessary to produce many copies of a particular protein, each of these nucleic acid therapeutic agents can affect a greater number of protein molecules through its indirect interaction than can a traditional macromolecular drug that relies on direct interaction with the targeted protein.
Nucleic acid therapeutic compounds may act in a number of different ways, but will most commonly fall into either one of two categories. The first category includes oligonucleotides that simulate or potentiate in some way a desired genetic effect. The activity stimulated by this type of nucleic acid therapeutic compound is commonly referred to as "gene therapy". The second category of nucleic acid therapeutic compounds includes inhibitory oligonucleotides wherein the nucleic acid therapeutic compound inhibits the production of undesired proteins. Antisense oligonucleotides form a subclass of inhibitory nucleic acid therapeutic compounds, although compounds commonly assigned to this subclass may not always act in a true "antisense" manner. In addition to these two categories of therapeutic oligonucleotides, it should also be noted that it is also possible for nucleic acid therapeutic compounds to interact directly with the target proteins in much the same way as traditional therapeutic drugs.
True antisense interactions involve the hybridization of complementary oligonucleotides (hence, the term "antisense") to their selected nucleic acid target (e.g., viral RNA or other undesired genetic messages) in a sequence specific manner such that the complex thus formed, either alone or in combination with other reagent(s) (e.g., enzymes, such as RNAse) can no longer function as a template for the translation of genetic information into proteins. Other inhibitory oligonucleotides have sequences that are not necessarily complementary to a target sequence, but, like antisense oligonucleotides, have the potential to interfere with the expression (e.g., replication and/or translation) of the undesired genetic material. An antisense oligonucleotide may be designed to interfere with the expression of foreign genes (e.g., viral genes, such as HIV) or with the aberrant expression of endogenous genes (e.g., a normal gene that is aberrantly expressed as a mutated oncogene). These undesired genetic messages are involved in many disease states, including viral infections and carcinomas. Inhibitory oligonucleotides raise the possibility of therapeutic arrest of a disease state at the early replication and expression stage, rather than attacking the resulting protein at a later stage of disease progression as in the manner of traditional drugs.
Oligonucleotides used in gene therapy are designed to provide an oligonucleotide, or synthetic gene, having a desired effect that is otherwise absent or impaired in a patient. Each gene normally present in a human body is responsible for the manufacture of a particular protein that contributes to either the structure or functioning of the body. If this gene is defective or absent, protein synthesis will be faulty or nonexistant, and a deformity or genetic disease will result. Incorporation of nucleic acid therapeutic compounds into the genetic material of a patient's cells can be accomplished through a vehicle, such as a retrovirus, thus enabling production of the needed protein.
Irrespective of whether nucleic acid therapeutic compounds are designed for gene therapy, antisense therapy, or any other situation where it is desired to affect proteins at a genetic or other level, the design of these synthetic oligonucleotides is a key to the level of success that can be achieved. Importantly, these oligonucleotides must ordinarily be modified in a manner that imparts nuclease resistance to the oligonucleotide such that they are capable of surviving in the presence of the various nucleases that are endogenous to a human or animal body. The same holds true for oligonucleotide probes employed in the analysis of serum samples, because the same exogenous nucleases present in the human body that can degrade unmodified therapeutic oligonucleotides are also present in human serum and can degrade unmodified oligonucleotide probes in these samples as well.
Specifically, unmodified (or "wild type") oligonucleotides are susceptible to nuclease degradation at both the 3'- and 5'-positions of the internucleotide bonds that link the individual nucleoside units together in the completed oligonucleotide. Consequently, attempts to impart nuclease resistance to therapeutic oligonucleotides have been directed to modification of this internucleotide linkage, with success having been achieved primarily with respect to modification of the "non-bridging" oxygen atoms in the naturally occurring phosphodiester linkage. (E.g., phosphorothioate-modified oligonucleotides having a single non-bridging oxygen substituted with a sulfur atom (U.S. Pat. No. 3,846,402) and phosphorodithioate-modified oligonucleotides having both non-bridging oxygen atoms substituted with sulfur atoms (U.S. Pat. No. 5,218,103). However, sulfur-containing oligonucleotides such as these are known to bind to proteins, resulting in a level of non-specific activity that may not be acceptable. Moreover, phosphorothioate-modified oligonucleotides are particularly susceptible to nuclease degradation at the 3'-position of the modified internucleotide bonds, especially by nucleases leaving a 5'-phosphate following cleavage of the internucleotide bond, due to the fact that only one of the "non-bridging" oxygen atoms in the phosphodiester bond is modified.
There are a number of currently available methods for oligonucleotide synthesis that can be employed to generate oligonucleotides having modified backbones. These methods involve either solution or solid-phase synthesis. The more traditional approach of solution-based synthesis requires relatively small amounts of mononucleotide synthon reagents and can provide significant quantities of the desired end-product. However, solution synthesis has its drawback in that it requires tedious isolation and purification of the intermediate product following each addition of a mononucleotide subunit. As a result, solution-based phosphotriester chemistry is not suitable for the practical synthesis of longer oligonucleotides (i.e., greater than 6 bases in length) required for use in nucleic acid therapeutics. In the case of solid-phase synthesis, the entire reaction sequence is carried out on a solid support with mononucleotide subunits being added sequentially to form a growing chain attached at one end to the polymeric support. Thus, the solid-phase method allows for easy separation of the reagents, with the only real drawback of this method being that it requires an excess of the mononucleotide synthon reagents (several times the amount required for solution synthesis) as well as other expensive reagents.
It would be desirable to have a non-sulfur-containing modified oligonucleotide of a length that would be suitable for use as a nucleic acid therapeutic compound or as a diagnostic probe and would have a sufficient number of modified linkages to impart nuclease resistance to the modified oligonucleotide. It would be further desirable to have a polymer-supported method for synthesis of such a non-sulfur-containing modified oligonucleotide. One non-sulfur-containing modification involves substitution of a P--C bond in place of the P--O linkage at the 3'-position of an unmodified phosphodiester bond to yield a 3'-carbon modified internucleotide linkage. Monomeric 3'methylene phosphonate nucleotides necessary as intermediates for solution-based preparation of this modified phosphodiester bond have been prepared using solution chemistry. See, for example, Albrecht et al., Tetrahedron, 40, 79-85 (1984); Albrecht et al., J. Amer. Chem. Soc., 92, 5511-5513 (1970); Morr et al., GBF Monogr. Ser., Chem. Syn. Mol. Biol., 8, 107-113 (1987).
Traditional phosphodiester methods of solution phase synthesis have resulted in the incorporation of these monomeric modified oligonucleotide subunits into fully modified ribonucleotide 3'-methylene phosphonate dimers and trimers. Jones et al., J. Amer. Chem. Soc., 92, 5510-5511 (1970, analog incorporated into dimer); Mazur et al., Tetrahedron, 40(20), 3949-3956 (1984) (analog incorporated into trimer). Furthermore, Morr et al., GBF Monogr. Ser., Chem. Syn. Mol. Biol., supra., have reported the synthesis of a modified deoxyribonucleotide 3'-methylene phosphonate dimer from the same monomeric 3'-methylene phosphonate nucleosides with subsequent incorporation of the modified dimer (containing a single modified internucleotide linkage between the two monomeric subunits) into a longer oligonucleotide. Heinemann, et al., Nucleic Acids Res., 19, 427 (1991). However, these procedures are far too laborious to be amenable to the large scale production of modified oligonucleotides.
The multiple 3'-carbon modifications necessary to impart nuclease resistance to an oligonucleotide have not been reported in deoxyoligonucleotides longer than a trimer, due to the inherent limitations of phosphotriester chemistry. Moreover, the solution-phase methodologies of the prior art cannot be applied to the more rapid and efficient polymer supported methodologies of oligonucleotide synthesis, because the phosphonate synthons used in the phosphotriester methods do not have sufficient coupling efficiencies to work effectively out of solution phase.
Therefore, it is an object of the present invention to provide monomeric oligonucleotide intermediates useful in the polymer-supported synthesis of 3'-carbon modified oligonucleotides.
It is a further object of the present invention to provide a polymer-supported method for synthesis of oligonucleotides having multiple 3'-carbon modifications.
It is a still further object of the present invention to provide oligonucleotides having at least one 3'-carbon modification useful in nucleic acid therapeutics and/or nucleic acid diagnostics.